The present research plan proposes to establish an experimental basis, to examine the direct and specific influence of adrenergic agents in vitro, known to induce changes in eye pressure in vivo, in the target cells, namely the ciliary epithelium, the tissue responsible for aqueous humor formation. Preliminary studies on an NEI Pilot Project award have demonstrated the feasibility of separating PE/NPE in culture. Using human and rabbit eyes of different ages and, if possible, of glaucoma tissues, it is intended to accomplish the following: (1) Enzymatic dissociation and further separation of NPE from PE cells by isopycnic centrifugation. (2) Growth optimization of NPE and PE in hormonally defined culture media. (3) Cellular cloning of NPE and PE cell. (4) Studies on transforming NPE. Screening, isolation and characterization of NPE transformed cells. (5) Criteria to define a number of morphological and biochemical markers to identify NPE and PE cells from other cell types in culture. (6) To investigate biochemical responses of NPE cells, namely protein phosphorylation and secretion, to external stimuli (beta-adrenergic agonists, blockers and cAMP analogs), since Preliminary Studies have shown that a small number of proteins are highly phophorylated in PE cells. This hormone-dependent activation can be antagonized by blocking agents such as Timolol of Propanolol. (7) Identification of the protein substrates highly phosphorylated under hormonal action, by two-dimensional gel electrophoresis and tryptic mapping analysis. Elucidation of the regulatory mechanisms of aqueous humor secretion should be very important in understanding more the functions of the cells involved and should help in the development of pharmacologic means for controlling aqueous humor formation.